[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-01-13 - 1995-02-24 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 (previous version) with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by Umweltministerium Baden-Württemberg

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C11-13 (branched) alkyl, salts with O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Molecular formula:

C27H60NPS2O2 C28H62NPS2O2 C29H64NPS2O2 (main component)

IUPAC Name:

Amines, C11-13 (branched) alkyl, salts with O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Test material form:

liquid

Details on test material:

Before Substance ID change known as CAS 71888-91-0 EC 276-159-7

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: NCTC National Collection of Type Cultures, Central Public Health Laboratory in London

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Wistar rat S9

Test concentrations with justification for top dose:

Maximum concentration: 5000 µg/plate, as stipulated by the guideline
at least 5 analyzable concentration, covering at least 2 logarithmic decades

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: good solubilizing properties

Controls

Untreated negative controls:

yes

Remarks:

aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: min. 48 h

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: 3 plates per concentration, two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

As set out in OECD TG 471, and as stipulated under REACH

Evaluation criteria:

A test item is considered positive if either a significant dose-dependent increase in the number of revertants or a significant and reproducible increase for at least one concentration of the test item is observed.
A significant effect is described as follows:
A test item is considered mutagenic if the number of revertants is increased twice (TA 100) or trice (TA98, TA1535, TA1537) over background.
A dose-dependent increase of the number of revertant is considered also as an indication of a mutagenic potential, even if the above-mentioned increase was not reached.
Commonly valid conditions for the assessment of the results are:
- normal background growth in all agar plates
- normal ranges of spontaneous revertant rates compared with negative control groups without metabolic activation

Ranges of spontaneous revertant rates:
TA98: 15 - 60
TA100: 75 - 200
TA1535: 3 - 37
TA1537: 4 - 31

Statistics:

Of each three plates per concentration mean and standard deviation was calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Evaporation from medium: unknown
- Precipitation: at doses >= 500 mg/plate

RANGE-FINDING/SCREENING STUDIES:
In all plates treated with the test item a normal backgroung growth was observed. Due to precipitation at doses >= 500 mg/plate, in the main experiment the following concentrations were tested: 500, 250, 50, 25, and 5 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: partially exceeded, but without relevance for validity of the experiment

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to the previous version of OECD TG 471 and is sufficiently documented, validity criteria were met. Only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible. In consequence, the results of this well-conducted study can be considered to be sufficiently reliable to assess the mutagenic potential of the test item. At none of the tested concentrations neither a significant and reproducible increase in revertants nor a dose-dependent increase of revertants at at least one concentration of the test item was observed. Hence, the test item does not need to be considered as mutagenic in bacteria.

Executive summary:

The study was conducted under GLP according to the previous version of OECD TG 471. S. typhimurium strains TA98, TA100, TA1535, TA1537 were exposed to various concentrations of the test item ranging from 2.5 - 5000 µg/plate in the plate incorporation method ±S9. Two independent experiments were performed.

No cytotoxicity was observed up to the limit dose, precipitation occurred >= 500 µg/plate. Positive and negative control gave the appropriate results. At none of the tested concentrations neither a significant and reproducible increase in revertants nor a dose-dependent increase of revertants at at least one concentration of the test item was observed. Hence, the test item does not need to be considered as mutagenic in bacteria.